# ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA BERKELEY · 2021 · $312,936

## Abstract

Project Summary
Protein degradation is tightly regulated by ATP-dependent compartmental proteases of the AAA+ family. The
major AAA+ protease in eukaryotic cells is the 26S proteasome, a 35-subunit complex that degrades proteins
marked with poly-ubiquitin chains and controls protein homeostasis as well as numerous vital processes.
Despite the proteasome’s great importance for cell viability, its detailed mechanisms for substrate selection
and processing, and in particular its regulation and fine-tuning, for instance by substrate-attached ubiquitin
chains, remain largely elusive. During the past granting period, we were able to significantly advance our
understanding of proteasome structure and function. We solved high-resolution structures of the substrate-
engaged proteasome at different stages of the ATP-hydrolysis cycle, established the first complete kinetic
picture of substrate degradation, revealed how major conformational changes of the proteasome are coupled
to individual steps of substrate processing, and uncovered how these conformational transitions are in part
regulated by interactions between proteasomal subcomplexes. Our biochemical tools, recombinant expression
systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous
outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S
proteasome and other AAA+ motors, and the regulation of pathways connected to the ubiquitin-proteasome
system. Especially our newly established single-molecule FRET-based assays allow unprecedented studies of
substrate interactions and progression through the proteasome regulatory particle, as well as the
conformational dynamics of the proteasome. Exciting preliminary data indicate that substrate-attached ubiquitin
chains affect the conformational switching, the kinetics of substrate engagement and degradation, and the
unfolding power of the proteasome depending on the chain length and linkage type. A primary goal is to
investigate how the proteasome utilizes its three main ubiquitin receptors and allosteric networks between
proteasomal subcomplexes to read out this “ubiquitin code” and fine-tune its activities. We will employ a
multidisciplinary approach that includes in-vitro biochemical, single-molecule, and atomic-resolution structural
studies. A pathway upstream of the 26S proteasome is the AAA+ protein unfoldase Cdc48 (p97/VCP in
human). In a new research direction, we will use fluorescence- and FRET-based assays combined with a
series of differentially ubiquitinated and labeled model proteins to investigate how Cdc48 in complex with its
adaptor Ufd1/Npl4 engages and unfolds its substrates, and how the dynamics of Cdc48-adaptor interactions
determine substrate delivery, unfolding, and deubiquitination. Besides advancing our general understanding of
ubiquitin-dependent protein unfolding and degradation, our research also has substantial medical relevance
and o...

## Key facts

- **NIH application ID:** 10298469
- **Project number:** 2R01GM094497-10
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Andreas Martin
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $312,936
- **Award type:** 2
- **Project period:** 2011-07-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10298469

## Citation

> US National Institutes of Health, RePORTER application 10298469, ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome (2R01GM094497-10). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10298469. Licensed CC0.

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